Cspg4-targeting humanized chimeric antigen receptor, immune effector cell expressing chimeric antigen receptor, and applications thereof

ABSTRACT

Related are a CSPG4-targeting humanized chimeric antigen receptor and applications thereof, comprising a humanized anti-CSPG4 binding domain, a hinge region, a transmembrane domain, and a signal transduction domain. The anti-CSPG4 binding domain comprises an anti-CSPG4 antibody or antigen binding part. Related are an immune effector cell expressing the CSPG4-targeting humanized chimeric antigen receptor and applications of the cell.

CROSS REFERENCE TO THE RELATED APPLICATIONS

This application is the national phase entry of InternationalApplication No. PCT/CN2021/120016, filed on Sep. 23, 2021, which isbased upon and claims priority to Chinese Patent Application No.202011013925.1, filed on Sep. 24, 2020, the entire contents of which areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy is namedGBZD010_Sequence_Listing.txt, created on Mar. 22, 2023, and is 25,460bytes in size.

TECHNICAL FIELD

The present disclosure relates to the field of biomedicine, andparticularly to a CSPG4-targeting humanized chimeric antigen receptor,immune effector cells expressing the CSPG4-targeting humanized chimericantigen receptor, and applications thereof.

BACKGROUND ART

Cancer is currently one of the diseases with the highest mortality inthe world, and in recent years, research on cancer therapy has alsoshifted from traditional surgery, radiotherapy, chemotherapy to targetedtherapy, immunotherapy and other precise therapies. The key to precisetherapy is the selection of effective and safe targets.

Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecularweight-melanoma-associated antigen (HMW-MAA) or melanoma-associatedchondroitin sulfate proteoglycan (MCSP), a member of the chondroitinsulfate proteoglycan family, was first found to be highly expressed inmelanoma. In later studies, it was found that CSPG4 was also highlyexpressed in other malignant solid tumors, such as brain glioblastoma,triple negative breast cancer, head and neck squamous cell carcinoma andmesothelioma. CSPG4 was also highly expressed on the surface ofcapillaries in solid tumors. In contrast, CSPG4 is poorly expressed innormal tissues and critical organs. In addition, CSPG4 can regulatecell-matrix interaction, activate downstream ERK, FAK and othersignaling pathways, and play an important role in tumor cellproliferation, migration, angiogenesis and metastasis.

Through genetically engineered T cells, chimeric antigen receptor—T celltherapy targets tumor cell surface antigens thereby killing tumor cells.Chimeric antigen receptor—T cell therapy has a greater targetingproperty than conventional surgery and chemoradiotherapy; compared withantibody therapy, it has stronger tumor-killing effect and bettercurative effect on malignant tumors. The success of this therapy inhematologic tumors also lays the foundation for its entry into the fieldof solid tumor therapy. In view of the high expression of CSPG4 on thesurface of malignant solid tumors, its low expression in normal tissues,and its important role in tumorigenesis and metastasis, this makes it agood target for chimeric antigen receptor—T cell therapy. TheCSPG4-targeting humanized chimeric antigen receptor was prepared andstructurally optimized in this disclosure.

SUMMARY OF THE INVENTION

The purpose of the present disclosure is to provide a CSPG4-targetinghumanized chimeric antigen receptor (CAR), immune effector cellsexpressing the CAR, and applications thereof in cancer diagnosis,treatment and prevention.

Specifically, the present disclosure provides the following technicalsolutions:

In one aspect, the present disclosure provides a CSPG4-targetinghumanized chimeric antigen receptor (CAR), the CAR comprises: ananti-CSPG4 binding domain, a hinge region, a transmembrane domain, and asignal transduction domain.

In one aspect, in the CAR of the present disclosure, wherein theanti-CSPG4 binding domain comprises an anti-CSPG4 antibody or antigenbinding part comprising a heavy chain CDR selected from amino acidsequences shown in SEQ ID NOS: 5-7 or any variant thereof, and/or alight chain CDR selected from amino acid sequences shown in SEQ ID NOS:10-12 or any variant thereof, characterized in that the anti-CSPG4binding domain is humanized.

The CAR according to any of the preceding aspects, wherein the anti-CSPG4 binding domain is humanized means that, the variable regionframework of the anti-CSPG4 binding domain is humanized.

The CAR according to any of the preceding aspects, wherein the variableregion framework of the anti-CSPG4 binding domain is humanized meansthat, the heavy chain variable region framework comprises a heavy chainFR selected from amino acid sequences shown in SEQ ID NOS: 16-19 or anyvariant thereof; and/or, the light chain variable region frameworkcomprises a light chain FR selected from amino acid sequences shown inSEQ ID NOS: 20-23 or any variant thereof.

The CAR according to any of the preceding aspects, wherein the variableregion framework of the anti-CSPG4 binding domain is humanized meansthat, the heavy chain variable region framework comprises a heavy chainFR1 selected from the amino acid sequence shown in SEQ ID NO: 16 or anyvariant thereof, a heavy chain FR2 selected from the amino acid sequenceshown in SEQ ID NO: 17 or any variant thereof, a heavy chain FR3selected from the amino acid sequence shown in SEQ ID NO: 18 or anyvariant thereof, a heavy chain FR4 selected from the amino acid sequenceshown in SEQ ID NO: 19 or any variant thereof; and/or a light chain FR1selected from the amino acid sequence shown in SEQ ID NO: 20 or anyvariant thereof, a light chain FR2 selected from the amino acid sequenceshown in SEQ ID NO: 21 or any variant thereof, a light chain FR3selected from the amino acid sequence shown in SEQ ID NO: 22 or anyvariant thereof, a light chain FR4 selected from the amino acid sequenceshown in SEQ ID NO: 23 or any variant thereof.

The CAR according to any of the preceding aspects, wherein theanti-CSPG4 binding domain comprises an anti-CSPG4 antibody or antigenbinding part comprising a heavy chain CDR1 selected from the amino acidsequence shown in SEQ ID NO: 5 or any variant thereof, a heavy chainCDR2 selected from the amino acid sequence shown in SEQ ID NO: 6 or anyvariant thereof, a heavy chain CDR3 selected from the amino acidsequence shown in SEQ ID NO: 7 or any variant thereof; and/or a lightchain CDR1 selected from the amino acid sequence shown in SEQ ID NO: 10or any variant thereof, a light chain CDR2 selected from the amino acidsequence shown in SEQ ID NO: 11 or any variant thereof, a light chainCDR3 selected from the amino acid sequence shown in SEQ ID NO: 12 or anyvariant thereof.

The CAR according to any of the preceding aspects, wherein theanti-CSPG4 binding domain comprising an anti-CSPG4 antibody or antigenbinding part comprising a heavy chain variable region sequence selectedfrom the amino acid sequence shown in SEQ ID NO: 4 or any variantthereof; and/or a light chain variable region sequence selected from theamino acid sequence shown in SEQ ID NO: 9 or any variant thereof.

The CAR according to any of the preceding aspects, wherein theanti-CSPG4 binding domain comprises an anti-CSPG4 single-chain antibody(scFv), preferably the amino acid sequence of the anti-C SPG4single-chain antibody is selected from amino acid sequences shown in SEQID NOS: 2, 14 or any variant thereof.

The CAR according to any of the preceding aspects, wherein the heavy andlight chains of the anti-CSPG4 single-chain antibody are operably linkedby a linker, preferably the linker comprises an amino acid sequenceselected from the amino acid sequence shown in SEQ ID NO: 15 or anyvariant thereof.

The CAR according to any of the preceding aspects, wherein thetransmembrane domain is selected from one or more of the α, β, ζ chainsof TCR, CD3γ, CD3δ, CD3ε, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22,CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB, CD152,CD154, PD-1, NKp44, NKp46 and NKG2D transmembrane domains; preferably,the transmembrane domain is selected from one or more of CD8α, CD8β,CD4, CD45, PD-1, CD154, CD28 transmembrane domains; preferably, thetransmembrane domain is selected from one or more of CD8α, CD28transmembrane domains; more preferably, the amino acid sequence of thetransmembrane domain is selected from the amino acid sequence of SEQ IDNO: 27 or any variant thereof.

The CAR according to any of the preceding aspects, wherein the hingeregion is selected from one or more of a CD8 extracellular hinge region,an IgG1 Fc CH2CH3 hinge region, an IgD hinge region, a CD28extracellular hinge region, an IgG4 Fc CH2CH3 hinge region and a CD4extracellular hinge region; preferably, the hinge region is a CD8α hingeregion; more preferably, the amino acid sequence of the hinge region isselected from the amino acid sequence of SEQ ID NO: 25 or any variantthereof.

The CAR according to any of the preceding aspects, wherein the signaltransduction domain is selected from one or more of TCRξ, FcRγ, FcRβ,CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278(ICOS), CD66d, DAP10,DAP12 and CD3ζ intracellular signal regions; preferably, the signaltransduction domain is selected from a CD3ζ intracellular signal region;more preferably, the amino acid sequence of the signal transductiondomain is selected from the amino acid sequence of SEQ ID NO: 33 or anyvariant thereof.

The CAR according to any of the preceding aspects, wherein the signaltransduction domain further comprises one or more costimulatory domains;preferably, the costimulatory domain is selected from one or more ofCARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, 4-1BB,CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, DAP12,LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, 4-1BBL and 2B4 intracellularsignal regions; preferably, the costimulatory domain is selected from4-1BB, CD134, CD28 and OX40 intracellular signal regions; preferably,the costimulatory domain is selected from one or more of 4-1BB and CD28intracellular signal domains; more preferably, the amino acid sequenceof the costimulatory domain is selected from the amino acid sequences ofSEQ ID NOS: 29, 31 or any variant thereof.

In one aspect, the present disclosure provides an isolated nucleic acidmolecule comprising a polynucleotide sequence encoding the CAR of any ofthe preceding aspects; preferably, the nucleotide sequence of thenucleic acid molecule is selected from the nucleotide sequences of SEQID NOS: 1, 13 or any variant thereof.

In one aspect, the present disclosure provides a nucleic acid constructcomprising the nucleic acid molecule of any of the previous aspects;preferably, the nucleic acid construct is a viral vector; morepreferably, the viral vector is one or more of a retroviral vector, alentiviral vector, an adenoviral vector, an adeno-associated viralvector.

In one aspect, the present disclosure provides a virus comprising thenucleic acid molecule of any of the preceding aspects, or comprising thenucleic acid construct of any of the preceding aspects; preferably, thevirus is one or more of a retrovirus, a lentivirus, an adenovirus, anadeno-associated virus.

In one aspect, the present disclosure provides uses of the CAR of any ofthe preceding aspects, the nucleic acid molecule of any of the precedingaspects, the nucleic acid construct of any of the preceding aspects, thevirus of any of the preceding aspects in the preparation of geneticallymodified immune cells of targeting CSPG4-expressing tumor cells.

In one aspect, the present disclosure provides an isolated host cellexpressing the CAR of any of the preceding aspects, or comprising theisolated nucleic acid molecule of any of the preceding aspects, orcomprising the nucleic acid construct of any of the preceding aspects,or comprising the virus of any of the preceding aspects; preferably, thehost cell is a mammalian cell; more preferably, the host cell is one ormore of a T cell, an NK cell, a γδT cell, an NKT cell, a macrophage orcell line, a PG13 cell line, a 293 cell line and cell lines derivedtherefrom; more preferably, the host cell is a T cell; most preferably,the host cell is a primarily cultured T cell.

The host cell according to any of the preceding aspects, the host cellfurther expresses other effector molecules including but not limited toone or more of cytokines, chemokines, another chimeric antigen receptor(CAR), chemokine receptors, siRNAs/shRNAs or sgRNAs knocking down orknocking out PD-1 expression or proteins blocking PD-L1, TCRs, andsafety switches.

The host cell according to any of the preceding aspects, wherein thecytokine is selected from one or more of TNF-α, TNF-β, VEGF, TPO, NGF-β,PDGF, TGF-α, TGF-β, IGF-I, IGF-II, EPO, M-CSF, IL-1, IL-1α, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-21, IL-25

LIF

FLT-3, interferon, angiostatin, thrombospondin, and endostatin.

The host cell according to any of the preceding aspects wherein thechemokine is selected from one or more of CCL1, CCL11, CCL12, CCL13,CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22,CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1,CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12,CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL9, CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3,FAM19A4, and FAM19A5.

The host cell according to any of the preceding aspects wherein thechemokine receptor is selected from one or more of CCR1, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, CCR8, CCRL1, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7,CXCR1, and CXCR2.

The host cell according to any of the preceding aspects wherein thesafety switch is selected from one or more of HSVTK, VZVTK, iCaspase-9,iCaspase-1, iCaspase-8, truncated EGFR, and RQR8.

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising one or more of the CAR of any of the precedingaspects, the nucleic acid molecule of the preceding aspects, the nucleicacid construct of the preceding aspects, the virus of the precedingaspects, the host cell of any of the preceding aspects, and apharmaceutically acceptable carrier.

Uses of the CAR of any of the preceding aspects, the nucleic acidmolecule of any of the preceding aspects, the nucleic acid construct ofany of the preceding aspects, the virus of any of the preceding aspects,the host cell of any of the preceding aspects, or the pharmaceuticalcomposition of any of the preceding aspects, in the preparation of amedicament; preferably, the medicament is used in diagnosing, treatingor preventing cancer related diseases.

Preferably, the medicament is used in diagnosing, treating or preventingtumors expressing CSPG4; more preferably, the medicament is used indiagnosing, treating or preventing one or more selected from braincancer, breast cancer, head and neck cancer, melanoma, mesothelioma;more preferably, the brain cancer is selected from one or more of brainglioblastoma, astrocytoma, meningioma, oligodendroglioma, glioma, thebreast cancer is triple negative breast cancer, the head and neck canceris head and neck squamous cell carcinoma.

Advantageous effects of the present disclosure include:

the CSPG4-targeting humanized chimeric antigen receptor of the presentdisclosure, as well as immune effector cells expressing the humanizedchimeric antigen receptor, can reduce the generation of immune rejectionand ensure better efficacy of the engineered CSPG4 chimeric antigenreceptor—T cell therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of CSPG4 in different tumor cell lines.

FIG. 2 shows the expression of CSPG4 in human brain glioma samples.

FIG. 3 shows the basic structure of CSPG4-targeting humanized chimericantigen receptors.

FIG. 4 shows the expression of the CSPG4-targeting humanized chimericantigen receptors on T cells.

FIG. 5A shows the killing effect on tumor cells by co-culture ofCSPG4-targeting chimeric antigen receptor—T cells with tumor cells asmeasured by flow cytometry.

FIG. 5B shows the killing effect on tumor cells by co-culture ofCSPG4-targeting chimeric antigen receptor—T cells with tumor cells,quantified by flow cytometry assay.

FIG. 6 shows cytokine levels upon tumor killed by the CSPG4-targetingchimeric antigen receptor—T cells.

FIG. 7 shows killing of brain gliomas in vivo by the CSPG4-targetinghumanized chimeric antigen receptor—T cells.

FIG. 8 shows that the CSPG4-targeting humanized chimeric antigenreceptor—T cells increase the survival rate of brain glioma model mice.

DETAILED DESCRIPTION I. Definitions

In this disclosure, the scientific and technical terms used herein havethe meanings commonly understood by those skilled in the art, unlessotherwise specified. Also, as used herein, protein and nucleic acidchemistry, molecular biology, cell and tissue culture, microbiology,immunology, and laboratory procedures used herein are terms and routineprocedures widely used in the corresponding fields. Meanwhile, in orderto better understand the present disclosure, definitions andexplanations of related terms are provided below.

It should also be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

As used herein and unless otherwise specified, the term “about” whenreferring to a measurable value such as an amount, time period, or thelike, is meant to encompass variations of ±10%, more preferably ±5%,even more preferably ±1%, and still more preferably ±0.1% from the givenvalue, so long as such variations are suitable for practicing thedisclosed methods.

As used herein, the term “activation” refers to the state of a T cellthat has been sufficiently stimulated to induce detectable cellproliferation. Activation may also be associated with induced cytokineproduction and detectable effector function. The terms “activated Tcells” and the like refer to T cells that undergo cell division.

As used herein, the term “antibody” refers to an immunoglobulin moleculethat specifically binds to an antigen. Antibodies can be intactimmunoglobulins derived from natural sources or from recombinantsources, and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies of the present disclosure can exist in a variety of formsincluding, but not limited to: polyclonal antibodies, monoclonalantibodies, Fv, Fab and F(ab)₂, etc. as well as single chain antibodiesand humanized antibodies (Harlow et al. 1999, using Antibodies: ALaboratory Manual, cold Spring Harbor Laboratory Press, NY; Harlow etal. 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York;Houston et al. 1988, Proc. Natl. Acad. Sci., USA 85: 5879-5883; Bird etal. 1988, Science 242: 423-426).

As used herein, an “antibody fragment” or “antigen binding fragment” ofan antibody refers to any portion of a full-length antibody that is lessthan full-length, but that comprises at least a portion of the variableregion (e.g. one or more CDR and/or one or more antibody binding sites)of the antibody that binds antigen, and thus retains binding specificityas well as at least a portion of the specific binding capacity of thefull-length antibody. Thus, an antigen-binding fragment refers to anantibody fragment that comprises an antigen-binding portion that bindsthe same antigen as the antibody from which the antibody fragment isderived. Antibody fragments include antibody derivatives produced byenzymatic treatment of full-length antibodies, as well as syntheticallyproduced derivatives, e.g. recombinantly produced derivatives.Antibodies include antibody fragments. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)2, single chain Fv(scFv), Fv, dsFv, diabodies, Fd and Fd′ fragments and other fragments,including modified fragments (see, e.g. Methods in Molecular Biology,Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprisemultiple chains linked together, for example by disulfide bonds and/orby peptide linkers. Antibody fragments generally comprise at least orabout 50 amino acids, and typically at least or about 200 amino acids.Antigen-binding fragments include any antibody fragment that, wheninserted into an antibody framework (e.g. by displacement of thecorresponding region), results in an antibody that immunospecificallybinds (i.e. exhibits a Ka of at least or at least about 107-108M-1) toan antigen. A “functional fragment” or “analog of an anti-CSPG4antibody” is a fragment or analog that prevents or substantially reducesthe ability of the receptor to bind a ligand or initiate signaltransduction. As used herein, functional fragments generally have thesame meaning as “antibody fragments”, and in the case of antibodies,fragments that prevent or substantially reduce the ability of thereceptor to bind a ligand or initiate signal transduction, e.g. Fv, Fab,F(ab′)2, and the like. An “Fv” fragment consists of a dimer of one heavychain variable domain and one light chain variable domain formed bynon-covalent association (VH-VL dimer). In this configuration, the threeCDRs of each variable domain interact to determine a target binding siteon the surface of the VH-VL dimer, as is the case with intactantibodies. The six CDRs together confer target binding specificity tothe intact antibody. However, even a single variable domain (or half ofan Fv comprising only three target-specific CDRs) may still have theability to identify and bind targets. As used herein, the term“framework” or “FR” residues refers to those variable domain residuesother than the hypervariable region residues defined herein as CDRresidues.

As used herein, “monoclonal antibody” refers to a population ofidentical antibodies, meaning that each individual antibody molecule inthe population of monoclonal antibodies is identical to another antibodymolecule. This property is in contrast to the property of a polyclonalpopulation of antibodies comprising antibodies having a plurality ofdifferent sequences. Monoclonal antibodies can be prepared by a numberof well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917;and Nelson et al. J Clin Pathol (2000), 53, 111-117). For example,monoclonal antibodies can be made by immortalizing B cells, for exampleby fusion with myeloma cells to produce hybridoma cell lines or byinfecting B cells with a virus, such as EBV. Recombinant techniques canalso be used to prepare antibodies from clonal populations of host cellsin vitro by transforming host cells with plasmids carrying artificialsequences encoding the nucleotides of the antibodies.

An antibody “heavy chain”, as used herein, refers to the larger of thetwo types of polypeptide chains present in their naturally occurringconformations in all antibody molecules.

An antibody “light chain”, as used herein, refers to the smaller of thetwo types of polypeptide chains present in their naturally occurringconformations in all antibody molecules, and kappa and lambda lightchains refer to the two major antibody light chain isotypes.

As used herein, the term “scFv” refers to a fusion protein comprising atleast one variable region antibody fragment comprising a light chain andat least one variable region antibody fragment comprising a heavy chain,wherein the light and heavy chain variable regions are linked via ashort, flexible polypeptide linker and are capable of being expressed asa single chain polypeptide, and wherein the scFv retains the specificityof the intact antibody from which it is derived. As used herein, unlessspecified, a scFv can have the VL and VH variable regions described ineither order (e.g. relative to the N-terminus and C-terminus of thepolypeptide), a scFv can comprise a VL-linker-VH or can comprise aVH-linker-VL.

As used herein, the term “gene synthesis” refers to production usingrecombinant DNA technology or obtained using synthetic DNA or amino acidsequence technology available and well known in the art.

As used herein, the term “antigen” or “Ag” is defined as a molecule thatelicits an immune response that may involve antibody production, oractivation of specific immunologically active cells, or both. Theskilled artisan will appreciate that any macromolecule, includingvirtually all proteins or peptides, can be used as an antigen. Inaddition, the antigen may be derived from recombinant or genomic DNA.The skilled person will understand any DNA-which comprises a nucleotidesequence or part of a nucleotide sequence encoding a protein thatelicits an immune response, thus encoding the term “antigen” as usedherein. Furthermore, those skilled in the art will appreciate that theantigen need not be encoded solely by the full-length nucleotidesequence of the gene. It will be readily apparent that the presentdisclosure includes, but is not limited to, the use of partialnucleotide sequences of more than one gene, and that these nucleotidesequences are arranged in different combinations to elicit a desiredimmune response. Furthermore, the skilled person will appreciate thatthe antigen need not be encoded by a “gene” at all. It will be readilyapparent that the antigen may be produced, synthesized or may be derivedfrom a biological sample. Such biological samples may include, but arenot limited to, tissue samples, tumor samples, cells, or biologicalfluids.

The term “anti-tumor effect”, as used herein, refers to a biologicaleffect that may be clearly indicated by a reduction in tumor volume, areduction in the number of tumor cells, a reduction in the number ofmetastases, an increase in life expectancy, or an improvement in variousphysiological symptoms associated with a cancerous condition. An“anti-tumor effect” may also be clearly indicated by the ability of thepeptides, polynucleotides, cells and antibodies of the presentdisclosure to prevent the occurrence of a tumor at a first location.

The term “cancer” as used herein is defined as a disease characterizedby rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the blood stream and lymphatic system to otherparts of the body. Examples of various cancers include, but are notlimited to, brain cancer (e.g. astrocytoma, meningioma,oligodendroglioma, glioma, etc.), breast cancer (e.g. triple negativebreast cancer), head and neck cancer (e.g. head and neck squamous cellcarcinoma), melanoma, mesothelioma, etc.

As used herein, the term “co-stimulatory ligand” includes a molecule onan antigen-presenting cell (e.g. APC, dendritic cell, B cell, etc.) thatspecifically binds to an associated costimulatory molecule on a T cell,thereby providing a signal that mediates a T cell response, includingbut not limited to proliferation, activation, differentiation, etc. inaddition to the primary signal provided by, for example, binding of theTCR/CD3 complex to a peptide-loaded MHC molecule. costimulatory ligandsmay include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86),PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L),intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G,MICA, MICB, HVEM, lymphotoxin β receptor, 3/TR6, ILT3, ILT4, HVEM,agonists or antibodies that bind to the Toll ligand receptor, andligands that specifically bind to CSPG4. costimulatory ligands alsoinclude, inter alia, antibodies that specifically bind to costimulatorymolecules present on T cells, such as, but not limited to, antibodiesthat specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, CSPG4 and CD83.

As used herein, the term “co-stimulatory molecule” refers to anassociated binding partner on a T cell that specifically binds to acostimulatory ligand, thereby mediating a costimulatory response of theT cell, such as but not limited to proliferation, including but notlimited to MHC-class I molecules, BTLA and Toll ligand receptors.

As used herein, the term “co-stimulatory signal” refers to a signalthat, in combination with a primary signal, such as a TCR/CD3 junction,results in up-or down-regulation of T cell proliferation and/or keymolecules.

“Encoding” refers to the use of a specific sequence of nucleotides in apolynucleotide such as a gene, cDNA, or mRNA as a template to synthesizethe inherent properties of other polymers and macromolecules inbiological processes that have any one of a defined sequence ofnucleotides (i.e. rRNA, tRNA, and mRNA) or a defined sequence of aminoacids and the biological properties resulting therefrom. Thus, a geneencodes a protein if transcription and translation of a mRNAcorresponding to that gene produces the protein in a cell or otherbiological system. A nucleotide sequence that is identical to a mRNAsequence and is generally provided in the Sequence Listing, and anon-coding strand that serves as a template for transcription of a geneor cDNA, can both be referred to as encoding a protein or other productof that gene or cDNA.

As used herein, the term “endogenous” refers to any substance from orproduced within an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any substance introducedfrom or produced outside of an organism, cell, tissue or system.

“expression” is defined as the transcription and/or translation of aparticular nucleotide sequence driven by its promoter.

As used herein, the term “nucleic acid construct” refers to a vectorcomprising a recombinant polynucleotide comprising expression controlsequences operably linked to a nucleotide sequence to be expressed. Thenucleic acid construct comprises sufficient cis-acting elements forexpression; other elements for expression may be supplied by the hostcell or in an in vitro expression system. Nucleic acid constructsinclude all those known in the art, such as cosmids, plasmids (e.g.naked or contained in liposomes), and viruses (e.g. lentiviruses,retroviruses, adenoviruses, and adeno-associated viruses) thatincorporate recombinant polynucleotides.

“Homologous” refers to sequence similarity or sequence identity betweentwo polypeptides or between two nucleic acid molecules. When a positionin two compared sequences is occupied by the same base or amino acidmonomer subunit, for example, if a position in each of two DNA moleculesis occupied by adenine, the molecules are homologous at that position.The percentage of homology between two sequences is a function of thenumber of matching or homologous positions shared by the two sequencesdivided by the number of positions compared×100. For example, twosequences are 60% homologous if 6 of the 10 positions in the twosequences are matched or homologous. By way of example, the DNAsequences ATTGCC and TATGGC share 50% homology. Typically, a comparisonis made when two sequences are aligned to give maximum homology.

As used herein, the term “immunoglobulin” or “Ig” is defined as a classof proteins that function as antibodies. Antibodies expressed by B cellsare sometimes referred to as BCR (B cell receptor) or antigen receptors.Five members included in this class of proteins are IgA, IgG, IgM, IgDand IgE. IgA are primary antibodies present in body secretions such assaliva, tears, breast milk, gastrointestinal secretions, and mucussecretions of the respiratory and genitourinary tracts. IgG is the mostcommon circulating antibody. IgM is the predominant immunoglobulinproduced in the primary immune response of most subjects. It is the mosteffective immunoglobulin in agglutination, complement fixation and otherantibody responses and is important in combating bacteria and viruses.IgD is an immunoglobulin without known antibody function, but can beused as an antigen receptor. IgE are immunoglobulins that mediateimmediate hypersensitivity after exposure to allergens by causingmediator release from mast cells and basophils.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or peptide that naturally occurs in a living animal isnot “isolated”, but the same nucleic acid or peptide that is partiallyor completely separated from coexisting materials in its natural stateis “isolated”. An isolated nucleic acid or protein can exist insubstantially purified form or, for example, can exist in a non-naturalenvironment, such as a host cell.

Unless otherwise specified, a “polynucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence encoding a protein or RNA may also includeintrons to the extent that the nucleotide sequence encoding the proteinmay include intron (s) in certain versions.

As used herein, the term “operably linked” refers to a functionallinkage between a regulatory sequence and a heterologous nucleic acidsequence, which results in expression of the latter. For example, afirst nucleic acid sequence is operably linked to a second nucleic acidsequence when the first nucleic acid sequence is in a functionalrelationship with the second nucleic acid sequence. For example, apromoter is operably linked to a coding sequence if the promoter affectsthe transcription or expression of the coding sequence. Generally,operably linked DNA sequences are contiguous, wherein two protein codingregions must be joined in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of tumorantigen is intended to indicate an abnormal level of tumor antigenexpression in cells from a disease area, such as a solid tumor within aparticular tissue or organ of a patient, relative to the expressionlevel of normal cells from the tissue or organ. Patients with solidtumors or hematological malignancies characterized by overexpression oftumor antigens can be determined by standard assays known in the art.

“Parenteral” administration of an immunogenic composition includes, forexample, subcutaneous (s. c), intravenous (i. v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

The terms “patient”, “subject”, “individual”, and the like are usedinterchangeably herein and refer to any animal or cell thereof, whetherin vitro or in situ, that is amenable to the methods described herein.In some non-limiting embodiments, the patient, subject or individual isa human.

The term “polynucleotide” as used herein is defined as a strand ofnucleotides. In addition, nucleic acids are multimers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. Those skilled in the art have the general knowledgethat nucleic acids are polynucleotides that can be hydrolyzed tomonomeric “nucleotides”. Monomeric nucleotides can be hydrolyzed tonucleosides. Polynucleotides as used herein include, but are not limitedto, all nucleic acid sequences obtained by any means available in theart, including, but not limited to, recombinant means, i.e. cloningnucleic acid sequences from recombinant libraries or cell genomes, usingcommon cloning techniques and PCRTM, etc. and synthetic means.

As used herein, the terms “peptide”, “polypeptide”, and “protein” areused interchangeably and refer to a compound consisting of amino acidresidues covalently linked by peptide bonds. The protein or peptide mustcomprise at least two amino acids, and there is no limitation on themaximum number of amino acids that can comprise the sequence of theprotein or peptide. Polypeptides include any peptide or proteincomprising two or more amino acids linked to each other by peptidebonds. As used herein, the term refers to short chains, which are alsocommonly referred to in the art as peptides, oligopeptides andoligomers; and longer chains, which are commonly referred to in the artas proteins, which are of many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, and the like. Polypeptides include natural peptides,recombinant peptides, synthetic peptides, or combinations thereof.

As used herein, the term “safety switch” refers to an engineered proteindesigned to prevent the potential toxicity of cell therapy or tootherwise prevent adverse effects. In some cases, safety switch proteinexpression is conditionally controlled to address safety issues oftransplanted engineered cells that have permanently incorporated a geneencoding a safety switch protein into their genome. Such conditionalregulation may be variable and may include control through smallmolecule-mediated post-translational activation and tissue-specificand/or on-time transcriptional regulation. The safety switch can mediateinduction of apoptosis, inhibition of protein synthesis, DNAreplication, growth arrest, transcriptional and post-transcriptionalgene regulation, and/or antibody-mediated depletion. In some cases, thesafety switch protein is activated by an exogenous molecule (e.g. aprodrug) that, when activated, triggers the treated cell to undergoapoptosis and/or cell death. Examples of safety switch proteins include,but are not limited to, HSVTK, VZVTK, iCaspase-9, iCaspase-1,iCaspase-8, truncated EGFR, RQR8, and the like. In this strategy, theprodrug administered in the event of an adverse event is activated bythe suicide gene product and kills the transduced cell.

As used herein, the term “promoter” is defined as a DNA sequencerequired to initiate specific transcription of a polynucleotidesequence, recognized by, or directed by the synthetic machinery of acell.

As used herein, the term “promoter/regulatory sequence” refers to anucleic acid sequence required for expression of a gene product operablylinked to a promoter/regulatory sequence. In some instances, thesequence may be a core promoter sequence, and in other instances, thesequence may also include enhancer sequences and other regulatoryelements required for expression of the gene product.Promoter/regulatory sequences may, for example, be sequences thatexpress a gene product in a tissue-specific manner.

A “constitutive” promoter is a nucleotide sequence that, when operablylinked to a polynucleotide encoding or specifying a gene product, causesthe gene product to be produced in a cell under most or all of thephysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence that, when operablylinked to a polynucleotide encoding or specifying a gene product, causesthe gene product to be produced in a cell substantially only when aninducer corresponding to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence that, whenoperably linked to a polynucleotide encoding or defined by a gene,results in the production of a gene product in a cell essentially aslong as the cell is of a tissue type corresponding to the promoter.

As used herein, “specific binding” or “immunospecifically binding” withrespect to an antibody or antigen-binding fragment thereof is usedinterchangeably herein and refers to the ability of an antibody orantigen-binding fragment to form one or more non-covalent bonds with thesame antigen through non-covalent interactions between the antibody andthe antibody binding site of the antigen. The antigen may be an isolatedantigen or present in a tumor cell. Typically, an antibody thatimmunospecifically binds (or specifically binds) to an antigen is onethat binds to the antigen with an affinity constant Ka of about or 1×10⁷M⁻¹ or 1×10⁸ M⁻¹ or more (or a dissociation constant (Kd) of 1×10⁻⁷M or1×10⁻⁸ M or less). Affinity constants can be determined by standardkinetic methods for antibody reactions, e.g. immunoassays, surfaceplasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol11: 54; Englebienne (1998) Analyst. 123: 1599), isothermal titrationcalorimetry (ITC), or other kinetic interaction assays known in the art(see, e.g. Paul, ed. Fundamental Immunology, 2nd ed. Raven Press, NewYork, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619, whichdescribes an exemplary SPR and ITC method for calculating the bindingaffinity of an antibody). Instruments and methods for detecting andmonitoring the rate of binding in real time are known and commerciallyavailable (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GEHealthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

As used herein, a “substantially purified” cell is a cell that issubstantially free of other cell types. Substantially purified cellsalso refer to cells that have been separated from other cell types withwhich they are normally associated in their naturally occurring state.In some instances, a substantially purified cell population refers to ahomogeneous cell population. In other instances, the term simply refersto a cell that has been separated from the cell with which it isnormally associated in its native state. In some embodiments, the cellsare cultured in vitro. In other embodiments, the cells are not culturedin vitro.

As used herein, the term “therapeutic” refers to both therapeutic and/orprophylactic. Therapeutic effects are achieved through inhibition,alleviation or eradication of a disease state.

As used herein, the term “therapeutically effective amount” refers tothe amount of the subject compound that will elicit the biological ormedical response of a tissue, system or subject that is being sought bythe researcher, veterinarian, medical doctor or other clinician. Theterm “therapeutically effective amount” includes the amount of acompound that: when administered, it is sufficient to prevent thedevelopment of, or alleviate to some extent, one or more of the signs orsymptoms of the disorder or disease being treated. The therapeuticallyeffective amount will vary depending on the compound, the disease andits severity, and the age, weight, etc. of the subject to be treated.

“treating” a disease, as a term used herein, refers to reducing thefrequency or severity of at least one sign or symptom of the disease ordisorder that a subject is experiencing.

As used herein, the terms “transfected” or “transformed” or “transduced”refer to the process by which exogenous nucleic acid is transferred orintroduced into a host cell. A “transfected” or “transformed” or“transduced” cell is a cell that has been transfected, transformed ortransduced with an exogenous nucleic acid. Such cells include primarysubject cells and progeny thereof.

As used herein, the term “vector” is a composition of matter thatincludes an isolated nucleic acid, and which can be used to deliver theisolated nucleic acid to the interior of a cell. Many vectors are knownin the art, including but not limited to linear polynucleotides,polynucleotides related to ionic or amphipathic compounds, plasmids andviruses. Thus, the term “vector” includes an autonomously replicatingplasmid or virus. The term should also be interpreted to includenon-plasmid and non-viral compounds that facilitate transfer of nucleicacids into cells, such as, for example, polylysine compounds, liposomes,and the like. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated viral vectors, retroviral vectors,and the like.

II. Detailed Description of the Embodiments

In one aspect, the present disclosure provides humanized chimericantigen receptors capable of targeting CSPG4, and immune effector cellsexpressing the CSPG4-targeting humanized chimeric antigen receptors; inspecific aspects, the immune effector cells expressing theCSPG4-targeting humanized chimeric antigen receptor are T cellsexpressing the CSPG4-targeting humanized chimeric antigen receptor.

In one aspect, the CSPG4-targeting humanized chimeric antigen receptor(CAR) of the present disclosure comprises: an anti-CSPG4 binding domain,a hinge region, a transmembrane domain and a signal transduction domain,the anti-CSPG4 binding domain comprises an anti-CSPG4 antibody orantigen binding part comprising a heavy chain CDR selected from aminoacid sequences shown in SEQ ID NOS: 5-7 or any variant thereof, and/or alight chain CDR selected from amino acid sequences shown in SEQ ID NOS:10-12 or any variant thereof, the anti-CSPG4 binding domain ishumanized.

The CAR according to any of the preceding aspects, wherein theanti-CSPG4 binding domain is humanized means that, the variable regionframework of the anti-CSPG4 binding domain is humanized.

The CAR according to any of the preceding aspect, wherein the variableregion framework of the anti-CSPG4 binding domain is humanized, meaningthat the heavy chain variable region framework comprises one or more ofthe following amino acid residues: the amino acid at position 3 is Q,the amino acid at position 5 is V, the amino acid at position 6 is Q,the amino acid at position 9 is S, the amino acid at position 16 is A,the amino acid at position 17 is S, the amino acid at position 20 is V,the amino acid at position 38 is R, the amino acid at position 40 is A,the amino acid at position 43 is Q, the amino acid at position 46 is E,the amino acid at position 48 is M, the amino acid at position 63 is G,the amino acid at position 65 is T, the amino acid at position 69 is V,the amino acid at position 73 is D, the amino acid at position 84 is S,the amino acid at position 85 is S, and the amino acid at position 87 isK, the amino acid at position 88 is A, the amino acid at position 93 isV, and the amino acid at position 108 is L; and/or the light chainvariable region framework comprises one or more of the following aminoacid residues: the amino acid at position 3 is V, the amino acid atposition 9 is L, the amino acid at position 10 is S, the amino acid atposition 12 is P, the amino acid at position 18 is P, the amino acid atposition 19 is A, the amino acid at position 21 is I, the amino acid atposition 37 is L, the amino acid at position 40 is P, the amino acid atposition 41 is G, the amino acid at position 42 is Q, the amino acid atposition 45 is Q, the amino acid at position 58 is V, the amino acid atposition 59 is D, the amino acid at position 63 is S, the amino acid atposition 74 is K, the amino acid at position 76 is S, the amino acid atposition 77 is R, and the amino acid at position 79 is E, the amino acidat position 80 is A, the amino acid at position 83 is V, the amino acidat position 85 is V, the amino acid at position 100 is Q, and the aminoacid at position 108 is T.

The CAR according to any of the preceding aspects, wherein the variableregion framework of the anti-CSPG4 binding domain is humanized meansthat, the heavy chain variable region framework comprises a heavy chainFR selected from amino acid sequences shown in SEQ ID NOS: 16-19 or anyvariant thereof; and/or, the light chain variable region frameworkcomprises a light chain FR selected from amino acid sequences shown inSEQ ID NOS: 20-23 or any variant thereof.

The CAR according to any of the preceding aspects, wherein the variableregion framework of the anti-CSPG4 binding domain is humanized meansthat, the heavy chain variable region framework comprises a heavy chainFR1 selected from the amino acid sequence shown in SEQ ID NO: 16 or anyvariant thereof, a heavy chain FR2 selected from the amino acid sequenceshown in SEQ ID NO: 17 or any variant thereof, a heavy chain FR3selected from the amino acid sequence shown in SEQ ID NO: 18 or anyvariant thereof, a heavy chain FR4 selected from the amino acid sequenceshown in SEQ ID NO: 19 or any variant thereof; and/or a light chain FR1selected from the amino acid sequence shown in SEQ ID NO: 20 or anyvariant thereof, a light chain FR2 selected from the amino acid sequenceshown in SEQ ID NO: 21 or any variant thereof, a light chain FR3selected from the amino acid sequence shown in SEQ ID NO: 22 or anyvariant thereof, a light chain FR4 selected from the amino acid sequenceshown in SEQ ID NO: 23 or any variant thereof.

The CAR according to any of the preceding aspects, wherein the antibodyor antigen binding part comprises a heavy chain CDR1 selected from theamino acid sequence shown in SEQ ID NO: 5 or any variant thereof, aheavy chain CDR2 selected from the amino acid sequence shown in SEQ IDNO: 6 or any variant thereof, a heavy chain CDR3 selected from the aminoacid sequence shown in SEQ ID NO: 7 or any variant thereof; and/or alight chain CDR1 selected from the amino acid sequence shown in SEQ IDNO: 10 or any variant thereof, a light chain CDR2 selected from theamino acid sequence shown in SEQ ID NO: 11 or any variant thereof, alight chain CDR3 selected from the amino acid sequence shown in SEQ IDNO: 12 or any variant thereof.

The CAR according to any of the preceding aspects, wherein theanti-CSPG4 binding domain comprising an anti-CSPG4 antibody or antigenbinding part comprising a heavy chain variable region sequence selectedfrom the amino acid sequence shown in SEQ ID NO: 4 or any variantthereof; and/or a light chain variable region sequence selected from theamino acid sequence shown in SEQ ID NO: 9 or any variant thereof.

The CAR according to any of the preceding aspects, wherein theanti-CSPG4 binding domain comprises an anti-CSPG4 single-chain antibody(scFv) wherein the linker connecting the light chain and the heavy chainis humanized, preferably the linker comprises an amino acid sequenceselected from the amino acid sequence shown in SEQ ID NO: 15 or anyvariant thereof.

The CAR according to any of the preceding aspects, wherein the anti-CSPG4 single-chain antibody is derived from a monoclonal antibody raisedagainst a fragment of the CSPG4 protein.

The CAR according to any of the preceding aspects, wherein the aminoacid sequence of the anti-CSPG4 single-chain antibody comprises aminoacid sequences selected from SEQ ID NOS: 2, 14 or any variant thereof.

In one aspect, a humanized anti-CSPG4 binding domain has at leastgreater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more sequence identity to the anti-CSPG4 single-chain antibodyportion of any of the preceding aspects.

The CAR according to any of the preceding aspects, the transmembranedomain is selected from one or more of the α, β, ζ chains of TCR, CD3γ,CD3δ, CD3ε, CD3ζ, CD4, CD5 ,CD8α, CD8β, CD9, CD16, CD22, CD27, CD28,CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, PD-1,NKp44, NKp46, and NKG2D transmembrane domains; preferably, thetransmembrane domain is selected from one or more of CD8α, CD8β, CD4,CD45, PD-1, CD154, CD28 transmembrane domains; more preferably, thetransmembrane domain is selected from one or more of CD8α, CD28transmembrane domains; more preferably, the amino acid sequence of thetransmembrane domain is selected from the amino acid sequence of SEQ IDNO: 27 or any variant thereof.

The CAR according to any of the preceding aspects, the hinge region isselected from one or more of a CD8 extracellular hinge region, an IgG1Fc CH2CH3 hinge region, an IgD hinge region, a CD28 extracellular hingeregion, an IgG4 Fc CH2CH3 hinge region and a CD4 extracellular hingeregion; preferably, the hinge region is a CD8α hinge region; morepreferably, the amino acid sequence of the hinge region is selected fromthe amino acid sequence of SEQ ID NO: 25 or any variant thereof.

The CAR according to any of the preceding aspects, the signaltransduction domain is selected from one or more of TCRξ, FcRγ, FcRβ,CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278(ICOS), CD66d, DAP10,DAP12 and CD3ζ intracellular signal regions; preferably, the signaltransduction domain is selected from a CD3ζ intracellular signal region;more preferably, the amino acid sequence of the signal transductiondomain is selected from the amino acid sequence of SEQ ID NO: 33 or anyvariant thereof.

The CAR according to any of the preceding aspects, the signaltransduction domain further comprises one or more costimulatory domains;preferably, the costimulatory domain is selected from one or more of thegroup consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54,CD83, OX40, 4-1BB, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1,CD278, DAP10, DAP12, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, 41BBL, and2B4 intracellular signal regions; more preferably, the costimulatorydomain is selected from 4-1BB, CD134, CD28 and OX40 intracellular signalregions; more preferably, the costimulatory signal domain is selectedfrom one or more of 4-1BB, CD28 intracellular signal domains; morepreferably, the amino acid sequence of the costimulatory domain isselected from the amino acid sequences of SEQ ID NOS: 29, 31 or anyvariant thereof.

In another aspect, the present disclosure also provides aCSPG4-targeting single-chain antibody (scFV); in particular, the scFv isderived from monoclonal antibodies raised against fragments of the CSPG4protein; more specifically, the amino acid sequence of the scFvcomprises amino acid sequences selected from SEQ ID NOS: 2, 14 or anyvariant thereof.

In one aspect, the gene encoding CSPG4-targeting scFV comprisesnucleotide sequences selected from SEQ ID NOS: 1, 13 or any variantthereof.

In one aspect, the present disclosure provides a nucleic acid moleculeencoding a CAR or scFV according to any of the preceding aspects;preferably, the nucleotide sequences of the nucleic acid moleculeencoding scFV are selected from SEQ ID NOS: 1, 13 or a nucleic acidmolecule having at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In one aspect, the present disclosure provides a nucleic acid constructcomprising the nucleic acid molecule of any of the previous aspects;preferably, the nucleic acid construct is a viral vector; morepreferably, the viral vector is one or more of a retroviral vector, alentiviral vector, an adenoviral vector.

In one aspect, the present disclosure provides a virus comprising thenucleic acid molecule of any of the preceding aspects, or comprising thenucleic acid construct of any of the preceding aspects; preferably, thevirus is one or more of a retrovirus, a lentivirus, an adenovirus, anadeno-associated virus.

In one aspect, the present disclosure provides an isolated host cellexpressing the CAR of any of the preceding aspects, or comprising thenucleic acid construct of any of the preceding aspects, or comprisingthe virus of any of the preceding aspects; preferably, the host cell isa mammalian cell; more preferably, the host cell is one or more of a Tcell, an NK cell, a γδT cell, an NKT cell, a macrophage or cell line, aPG13 cell line, a 293 cell line and cell lines derived therefrom; morepreferably, the host cell is a T cell; most preferably, the host cell isa primarily cultured T cell.

In one aspect, the host cell further expresses other effector moleculesincluding but not limited to one or more of cytokines, chemokines,another chimeric antigen receptor (CAR), chemokine receptors,siRNA/shRNAs or sgRNAs knocking down or knocking out PD-1 expression orproteins blocking PD-L1, TCRs, and safety switches; preferably, thecytokine is selected from one or more of TNF-α, TNF-β, VEGF, TPO, NGF-β,PDGF, TGF-α, TGF-β, IGF-I, IGF-II, EPO, M-CSF, IL-1, IL-1α, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-21, IL-25

LIF

FLT-3, interferon, angiostatin, thrombospondin, and endostatin;preferably, the chemokine is selected from one or more of CCL1, CCL11,CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL 18, CCL19, CCL2, CCL20,CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3,CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL3,CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCL8, XCL1, XCL2, FAM19A1, FAM19A2,FAM19A3, FAM19A4 and FAM19A5. Preferably, the chemokine receptor isselected from one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7,CCR8, CCRL1, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CXCR1, CXCR2;preferably, the safety switch is selected from one or more of HSVTK,VZVTK, iCaspase-9, iCaspase-1, iCaspase-8, truncated EGFR and RQR8.

In one aspect, the present disclosure provides an immune effector cellthat expresses the CSPG4-targeting chimeric antigen receptor; inparticular, the present disclosure provides a T cell expressing theCSPG4-targeting chimeric antigen receptor (CAR-T); more specifically,the T cell is an activated T cell; more specifically, the activation ofthe T cells is accomplished by stimulation with CD3 and/or CD28antibodies.

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising one or more of the CAR, the nucleic acidmolecule, the nucleic acid construct, the virus, the cell of any of thepreceding aspects, and a pharmaceutically acceptable carrier.

It will be appreciated that therapeutic agents according to theembodiments will be administered with suitable pharmaceuticallyacceptable carriers, excipients, and other agents incorporated intoformulations to provide improved transfer, delivery, tolerability, andthe like. A large number of suitable formulations can be found in allpharmacopoeias known to pharmaceutical chemists: Remington'sPharmaceutical Science (15^(th) edition, Mack Publishing Company,Easton, Pa. (1975)), in particular Chapter 87 of Blaug, Seymour therein.These formulations include, for example, powders, pastes, ointments,gels, waxes, oils, lipids, lipid-containing (cationic or anionic)carriers (e.g. Lipofectin™), DNA conjugates, anhydrous syrups,oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing polyethylene glycol. Any of theforegoing mixtures may be suitable for the treatment or therapyaccording to the present disclosure, provided that the activeingredients in the formulation are not inactivated by the formulationand that the formulation is physiologically compatible and tolerates theroute of administration.

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion medium, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, which are compatible with pharmaceuticaladministration. Suitable pharmaceutically acceptable carriers aredescribed in the latest edition of Remington's Pharmaceutical Sciences,a standard reference text in the art, which is incorporated herein byreference. Preferred examples of such carriers or diluents include, butare not limited to, water, saline, ringer's solution, dextrose solution,and 1-10% human serum albumin. Liposomes and non-aqueous vehicles suchas fixed oils can also be used. The use of such medium and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional medium or agent is incompatible with theantibody, its use in the compositions is contemplated.

Further, a pharmaceutical composition provided by the present disclosurecan be used to treat diseases associated with inflammatory factors suchas cancer, inflammatory diseases, autoimmune diseases, etc.

In one aspect, the use of a CAR, a nucleic acid molecule, a nucleic acidconstruct, a virus, a cell, a pharmaceutical composition of any of thepreceding aspects in the preparation of a medicament for diagnosing,treating or preventing cancer.

In one embodiment, the medicament may be used as a therapeutic agent.Such agents will typically be used to diagnose, treat, ameliorate,and/or prevent a disease or pathology associated with aberrant CSPG4expression, activity, and/or signal transduction in a subject. Treatmentregimens can be performed using standard methods by identifying asubject, e.g. a person patient having (or at risk of or developing) adisease or disorder associated with aberrant CSPG4 expression, activity,and/or signal transduction, e.g. cancer or other neoplastic disorder. Anantibody or antibody fragment specifically binding to a target antigen(such as CSPG4) is used as a chimeric antigen receptor (CAR) of thepresent disclosure, and the disclosed CAR is preferably prepared by anantibody having high specificity and high affinity to the target antigen(such as CSPG4), preferably, the prepared CAR is further used togenetically modify immune cells (such as T cells). By administering CARand/or genetically modified immune cells of the present disclosure,expression, activity, and/or signal transduction functions of a target(e.g. CSPG4) can be eliminated, inhibited, or prevented. Binding of atarget (e.g. CSPG4) to an endogenous ligand to which it naturally bindscan be eliminated or inhibited or prevented by administration of a CARand/or genetically modified immune cell of the present disclosure, e.g.CSPG4 expression, activity and/or signal transduction can be modulated,blocked, inhibited, reduced, antagonized, neutralized, or otherwiseprevented following administration of a CAR and/or genetically modifiedimmune cell of the present disclosure. In some embodiments, to treat adisease or disorder associated with aberrant CSPG4 expression,antibodies or antibody fragments of the anti-CSPG4 antibody heavy andlight chain CDR can be prepared into the disclosed CAR and/orgenetically modified immune cells for administration to a subject. Inone embodiment, the disease or disorder associated with aberrant CSPG4expression may be cancer.

For purposes of clarity and conciseness, features are described hereinas part of the same or separate embodiments, however, it will beunderstood that the scope of the disclosure may include some embodimentshaving a combination of all or some of the features described.

EXAMPLES Example 1: Detection of CSPG4 Expression in Tumor Cell Lines

To determine the expression of CSPG4 antigen in tumors, tumor cell linesof melanoma (WM-266-4 cell line), glioma (LN-229, U87-MG cell line) andbreast cancer (MDA-MB-231 cell line) were first selected for detection.Meanwhile, in order to further verify the expression of CSPG4 in primarybrain glioma, sections containing brain glioma tissues of multiplepatients as well as normal tissues were detected by animmunohistochemical method using CSPG4 antibody.

Method: tumor cells were collected and washed three times with 1×PBS,then stained with CSPG4 antibody on ice for 30 min, after washing with1×PBS, APC-conjugated secondary antibody was added, incubated at roomtemperatures for 20 min, washed with 1×PBS, and CSPG4 expression ontumor cells was detected by flow cytometry.

Immunohistochemical staining: paraffin sections were deparaffinized withxylene and rehydrated with alcohol, followed by antigen retrieval withsodium citrate, then blocked in serum for 1 h. The CSPG4 antibody wasstained overnight at 4° C., and then incubated with horseradishperoxidase-coupled secondary antibody for 30 min. After washing, it wasdeveloped with DAB and then stained with hematoxylin. Sections wereobserved and photographed under a microscope after staining wascompleted.

Results: as can be seen from FIG. 1 , the detected tumor cells showedhigh expression of CSPG4 in all cases.

As can be seen from FIG. 2 , the expression of CSPG4 was detected tovarying degrees in the brain glioma samples of all patients, while theexpression of CSPG4 was not found in normal brain tissues. This suggeststhat CSPG4 is a better target for brain glioma therapy.

Example 2: Cell Culture

Cell lines used in the disclosure include 293T cells cultured in IMDMmedium containing 10% FBS, 1% glutamax, 1% two types of antibiotics.WM-266-4, LN-229, U87-MG and MDA-MB-231 cell culture medium were 10% FBSin RPMI640 or DMEM. GFP positive cells were prepared by infection ofretrovirus encoding GFP and tumor cell lines for mice were transfectedwith lentivirus or retrovirus encoding luciferase or GFP-fireflyluciferase (GFP-FFLuc).

Example 3: Construction of Chimeric Antigen Receptors

Existing chimeric antigen receptors for CSPG4 are derived from the mousemonoclonal antibody 763.74 (WO2015080981), however, considering thatmouse antibodies or chimeric antigen receptors will elicit “humananti-mouse” antibodies in human bodies, thereby greatly diminishing thetherapeutic efficacy of the antibody or chimeric antigen receptor—Tcells. In view of the above considerations, the present disclosurehumanizes the chimeric antigen receptor derived from the antibody763.74, and also optimizes the structure of the original chimericantigen receptor (the original mouse chimeric antigen receptor (mCSPG4)is a heavy chain-light chain structure, and the humanized chimericantigen receptor has two structures of a heavy chain-light chain(hCSPG4-HL) and a light chain-heavy chain (hCSPG4-LH), see FIG. 3 ), andthe present disclosure also makes a functional comparison with the mousechimeric antigen receptor.

The humanized anti-CSPG4 chimeric antigen receptors of the presentdisclosure consists of a single-chain antibody region, a CD8α hingeregion, a CD8α transmembrane region, a CD28 or 4-1BB costimulatoryfactor region, and a CD3ζ signal transduction region. The single-chainantibody region of the chimeric antigen receptor is derived from thehumanization of the sequence of the antibody 763.74. Chimeric antigenreceptors are generated by gene synthesis and then cloned intoretroviral vectors. The correctness of the sequence was confirmed bysequencing after cloning.

TABLE 1 Sequence List:SEQ ID NO: 1: nucleic acid sequence of the humanized CSPG4-HL (hCSPG4-HL) single-chainantibody regionATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGACAGGTTCAGCTGGTGCAGTCTGGCAGCGAGCTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTAGCGGCTACACATTCACCGACTACAGCATGCACTGGGTCCGAAAGGCCCCTGGACAGGGACTTGAATGGATGGGCTGGATCAACACCGCCACAGGCGAGCCTACCTACGCCGATGGCTTCACAGGCAGATTCGTGATCAGCCTGGACACCAGCGCCAGAACCGTGTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCGTGTACTTCTGCTTCAGCTACTACGACTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGACATCGTGCTGACACAGAGCCCTCTGAGCCTGCCTGTTACACCTGGCGAGCCTGCCAGCATCAGCTGTAGAGCCAGCCAGACCATCTACAAGAACCTGCACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATTAAGTACGGCAGCGACAGCATCTCCGGCGTGCCAGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTACACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCTACAGCACCCCTTGGACATTCGGCCAGGGCACCAAGCTGGAAATCAAGSEQ ID NO: 2: protein sequence of the humanized CSPG4-HL (hCSPG4-HL) single-chainantibody regionMEFGLSWLFLVAILKGVQCSRQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRKAPGQGLEWMGWINTATGEPTYADGFTGRFVISLDTSARTVYLQISSLKAEDTAVYFCFSYYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPLSLPVTPGEPASISCRASQTIYKNLHWYLQKPGQSPQLLIKYGSDSISGVPDRFSGSGSGTDYTLKISRVEAEDVGVYYCLQGYSTPWTFGQGTKLEIKSEQ ID NO: 3: nucleic acid sequence of the humanized CSPG4 (hCSPG4) single-chainantibody heavy chainCAGGTTCAGCTGGTGCAGTCTGGCAGCGAGCTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTAGCGGCTACACATTCACCGACTACAGCATGCACTGGGTCCGAAAGGCCCCTGGACAGGGACTTGAATGGATGGGCTGGATCAACACCGCCACAGGCGAGCCTACCTACGCCGATGGCTTCACAGGCAGATTCGTGATCAGCCTGGACACCAGCGCCAGAACCGTGTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCGTGTACTTCTGCTTCAGCTACTACGACTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCTSEQ ID NO: 4: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodyheavy chain QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRKAPGQGLEWMGWINTATGEPTYADGFTGRFVISLDTSARTVYLQISSLKAEDTAVYFCFSYYDYWGQGTLVTVSSSEQ ID NO: 5: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodyheavy chain CDR1 GYTFTDYSSEQ ID NO: 6: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodyheavy chain CDR2 INTATGEPSEQ ID NO: 7: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodyheavy chain CDR3 FSYYDYSEQ ID NO: 8: nucleic acid sequence of the humanized CSPG4 (hCSPG4) single-chainantibody light chainGACATCGTGCTGACACAGAGCCCTCTGAGCCTGCCTGTTACACCTGGCGAGCCTGCCAGCATCAGCTGTAGAGCCAGCCAGACCATCTACAAGAACCTGCACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATTAAGTACGGCAGCGACAGCATCTCCGGCGTGCCAGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTACACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCTACAGCACCCCTTGGACATTCGGCCAGGGCACCAAGCTGGAAATCAAGSEQ ID NO: 9: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chainDIVLTQSPLSLPVTPGEPASISCRASQTIYKNLHWYLQKPGQSPQLLIKYGSDSISGVPDRFSGSGSGTDYTLKISRVEAEDVGVYYCLQGYSTPWTFGQGTKLEIKSEQ ID NO: 10: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chain CDR1 RASQTIYKNLHSEQ ID NO: 11: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chain CDR2 YGSDSISSEQ ID NO: 12: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chain CDR3 LQGYSTPWTSEQ ID NO: 13: nucleic acid sequence of the humanized CSPG4-LH (hCSPG4-LH)single-chain antibody regionATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGAGACATCGTGCTGACACAGAGCCCTCTGAGCCTGCCTGTTACACCTGGCGAGCCTGCCAGCATCAGCTGTAGAGCCAGCCAGACCATCTACAAGAACCTGCACTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATTAAGTACGGCAGCGACAGCATCTCCGGCGTGCCAGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTACACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTCTGCAAGGCTACAGCACCCCTTGGACATTCGGCCAGGGCACCAAGCTGGAAATCAAGGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTTCAGCTGGTGCAGTCTGGCAGCGAGCTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTAGCGGCTACACATTCACCGACTACAGCATGCACTGGGTCCGAAAGGCCCCTGGACAGGGACTTGAATGGATGGGCTGGATCAACACCGCCACAGGCGAGCCTACCTACGCCGATGGCTTCACAGGCAGATTCGTGATCAGCCTGGACACCAGCGCCAGAACCGTGTACCTGCAGATCAGCTCTCTGAAGGCCGAGGATACCGCCGTGTACTTCTGCTTCAGCTACTACGACTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCTSEQ ID NO: 14: protein sequence of the humanized CSPG4-LH (hCSPG4-LH) single-chainantibody regionMEFGLSWLFLVAILKGVQCSRDIVLTQSPLSLPVTPGEPASISCRASQTIYKNLHWYLQKPGQSPQLLIKYGSDSISGVPDRFSGSGSGTDYTLKISRVEAEDVGVYYCLQGYSTPWTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRKAPGQGLEWMGWINTATGEPTYADGFTGRFVISLDTSARTVYLQISSLKAEDTAVYFCFSYYDYWGQGTLVTVSSSEQ ID NO: 15: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylinker GGGGSGGGGSGGGGSGGGGSSEQ ID NO: 16: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodyheavy chain FR1 QVQLVQSGSELKKPGASVKVSCKASSEQ ID NO: 17: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodyheavy chain FR2 MHWVRKAPGQGLEWMGWSEQ ID NO: 18: protein sequence of the humanized CSPG4L (hCSPG4) single-chain antibodyheavy chain FR3 TYADGFTGRFVISLDTSARTVYLQISSLKAEDTAVYFCSEQ ID NO: 19: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodyheavy chain FR4 WGQGTLVTVSSSEQ ID NO: 20: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chain FR1 DIVLTQSPLSLPVTPGEPASISCSEQ ID NO: 21: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chain FR2 WYLQKPGQSPQLLIKSEQ ID NO: 22: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chain FR3 GVPDRFSGSGSGTDYTLKISRVEAEDVGVYYCSEQ ID NO: 23: protein sequence of the humanized CSPG4 (hCSPG4) single-chain antibodylight chain FR4 FGQGTKLEIKSEQ ID NO: 24: nucleic acid sequence of CD8α hinge regionACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATSEQ ID NO: 25: protein sequence of CD8α hinge regionTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSEQ ID NO: 26: nucleic acid sequence of CD8α transmembrane regionATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCSEQ ID NO: 27: protein sequence of CD8α transmembrane regionIYIWAPLAGTCGVLLLSLVITLYCSEQ ID NO: 28: nucleic acid sequence of CD28 costimulatory factorAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC TATCGCTCCSEQ ID NO: 29: protein sequence of CD28 costimulatory factorRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSSEQ ID NO: 30: nucleic acid sequence of 4-1BB costimulatory factorAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGSEQ ID NO: 31: protein sequence of 4-1BB costimulatory factorKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSEQ ID NO: 32: nucleic acid sequence of CD3ζ signal transduction regionAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCSEQ ID NO: 33: protein sequence of CD3ζ signal transduction regionRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO. 34: nucleic acid sequence of CD19-CARATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGAGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTGCAGCTGCAGCAGTCTGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGGACCTCAGTCACCGTCTCCTCAACGCGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCSEQ ID NO. 35: protein sequence of CD19-CARMEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 4: Preparation of the Retroviruses for Chimeric AntigenReceptors

Murine leukemia retrovirus (MMLV) is prepared by 293T cells transientlytransfected using a three-plasmid system (GFS-CAR, MMLV-gag-pol andRV-RD). CD19-CAR was used as a control for subsequent studies, whilemurine (mCSPG4) and humanized (hCSPG4-HL, hCSPG4-LH) CARs were thetarget CARs to be tested. At 48 and 72 h after transfection, 293T cellsupernatants were harvested for infection of T cells. Among them, thenucleotide sequence of CD19-CAR is shown in SEQ ID NO: 34, and the aminoacid sequence thereof is shown in SEQ ID NO: 35.

Example 5: Preparation of T Cells Expressing CSPG4-targeting HumanizedChimeric Antigen Receptor 1. Infection and Amplification of T Cells

Blood donated from healthy individuals was used to isolate peripheralblood mononuclear cells. Mononuclear cells were isolated usinglymphocyte density gradient separation. After activation of the isolatedmononuclear cells by antibodies to CD3 and CD28, T cells were infectedwith the harvested retroviruses. The chimeric antigen receptortransfected T cells were then amplified for detection of chimericantigen receptor expression and tumor killing.

2. Detection of the Expression of the CSPG4 Chimeric Antigen Receptors

A portion of amplified chimeric antigen receptor—T cells were harvestedand the expression of CSPG4 chimeric antigen receptors were detectedusing biotin-labeled protein L, and the secondary antibody wasphycoerythrin-labeled avidin. Flow cytometry was used to detect theexpression of the chimeric antigen receptors on T cells.

Results: as can be seen from FIG. 4 , the murine (mCSPG4) or humanized(hCSPG4-HL or hCSPG4-LH) CSPG4 chimeric antigen receptors (CD28 or 4-1BBcostimulatory domain) can be expressed on human T cells. The expressionof murine chimeric antigen receptors was slightly higher than that ofhumanized receptors, mainly due to the decreased binding of the stainingreagent caused by the humanization of the chimeric antigen receptor.

Example 6: Killing Effect of CSPG4 Humanized Chimeric Antigen Receptor—TCells on Tumor Cells (Cell Co-culture)

To compare whether the engineered chimeric antigen receptor of thepresent disclosure can mediate T cell killing of tumor cells highlyexpressing CSPG4 as the original murine receptor, co-culture experimentswere designed to test the killing of various tumor cells by threechimeric antigen receptor—T cells (mCSPG4, hCSPG4-HL, hCSPG4-LH). Aftertumor cell attachment, T cells expressing control (chimeric antigenreceptor against CD19, CD19-CAR), murine and two humanized CSPG4chimeric antigen receptors were added, and the killing thereof onmelanoma, glioma and breast cancer cells was detected by flow cytometry.

Method: GFP-labeled tumor cells were seeded at a density of 1-2×10⁵ perwell in 24-well plates, and 24 h later corresponding numbers of T cellswere added at different ratios. After 7 days the cells in the wells wereharvested and the remaining cells were detected by flow cytometry. Tcells were detected using CD3, and tumor cells were detected using GFP.

Results: as shown in FIGS. 5A and 5B, murine CSPG4 chimeric antigenreceptor—T cells show some killing effect on three tumor cells. Incontrast, the T cells of the humanized CSPG4 chimeric antigen receptorsof the present disclosure can kill tumor cells more effectively inco-culture with three tumor cells, and in particular, thestructurally-optimized humanized CSPG4 chimeric antigen receptor(hCSPG4-LH) can completely eliminate tumor cells.

Example 7: Effect of CSPG4 Humanized Chimeric Antigen Receptor—T Cellson Cytokine Release During Tumor Cell Killing

Method: enzyme-linked immunosorbent assay (ELISA)

Supernatants were harvested 24 h after the addition of T cells inexperiments where tumor cells were co-cultured with chimeric antigenreceptor—T cells. Cytokines were detected in the supernatants using anELISA kit.

Results: the results are consistent with that of the cytotoxicityexperiments, it can be seen from FIG. 6 that the T cells of thehumanized CSPG4 chimeric antigen receptor engineered and optimized inthe present disclosure released higher levels of cytokines duringkilling compared to the lower Th1-type cytokines (IFN-gamma, IL-2)released by the murine receptor, indicating that the humanizationengineered and structurally-optimized CSPG4 chimeric antigen receptorhave better killing effect on tumors with high CSPG4 expression than theoriginal murine receptor.

Example 8: Survival Experiments of Xenografted Mice

To determine the tumor killing effect of the T cells of the CSPG4humanized chimeric antigen receptor engineered and optimized in thepresent disclosure in vivo, a mice brain glioma model was selected.Luciferase-labeled tumor cells were injected intracerebrally into nudemice, control (CD19-targeting chimeric antigen receptor) andCSPG4-targeting humanized chimeric antigen receptor T cells wereinjected intraventricularly after tumor formation, and tumor clearanceby T cells and survival of treated mice were detected by imaging.

Method: the in vivo experiment of CSPG4 humanized chimeric antigenreceptor—T cells targeting to brain gliomas were performed in nude miceat 6-8 weeks. GFP-FFLuc labeled tumor cell lines were stereotaxicallyimplanted in the brain of nude mice, and 2-3 weeks later, oncesuccessful tumor implantation was confirmed in an in vivo imagingsystem, control or CSPG4-targeting humanized chimeric antigen receptor—Tcells were injected intraventricularly. Tumor killing by T cells wasobserved by weekly imaging.

In vivo survival experiments, GFP-FFLuc-labeled LN-229 cells werestereotaxically injected into the brain of mice, 44 days later, thesolvent PBS, control CD19-CAR T cells and CSPG4-targeting mouse (mCSPG4)and humanized (hCSPG4-HL, hCSPG4-LH) CAR-T cells were intraventricularlyinjected. The body weight of the mice was measured weekly thereafter andthe time of death of the mice was recorded.

Results: as shown in FIG. 7 , the T cells of the CSPG4 chimeric antigenreceptors engineered and optimized in the present disclosure can cleartumors in most mice and control tumor recurrence within a certain periodof time, as compared to the continued tumor growth of the control group.This result indicates that T cells of CSPG4-targeting humanized chimericantigen receptors can kill tumors and control tumor recurrence in vivo.

Survival of LN-229 brain glioma mice undergoing control and T celltherapy of CSPG4-targeting chimeric antigen receptors was also detected.As shown in FIG. 8 , compared with control PBS and CD19-CAR-T, murineCSPG4 CAR-T (mCSPG4) can prolong the survival time of mice to a certainextent, while the humanized CSPG4 CAR-T of the present disclosure(hCSPG4-HL and hCSPG4-LH) achieves better therapeutic effect than mouseCAR-T. In particular, the structurally optimized hCSPG4-LH CAR-Tsignificantly prolongs the survival time of mice. This demonstrates thatthe humanized CSPG4 CAR-T of the present disclosure can exhibit bettertherapeutic effect on tumors than the original mouse CAR-T.

What is claimed is:
 1. A CSPG4-targeting humanized chimeric antigenreceptor (CAR) comprising: an anti-CSPG4 binding domain, a hinge region,a transmembrane domain and a signal transduction domain, the anti-CSPG4binding domain comprises an anti-CSPG4 antibody or antigen binding partcomprising a heavy chain CDR selected from amino acid sequences shown inSEQ ID NOS: 5-7 or any variant thereof, and/or a light chain CDRselected from amino acid sequences shown in SEQ ID NOS: 10-12 or anyvariant thereof, characterized in that the anti-CSPG4 binding domain ishumanized.
 2. The humanized chimeric antigen receptor (CAR) of claim 1,wherein the anti-CSPG4 binding domain is humanized means that, thevariable region framework of the anti-CSPG4 binding domain is humanized.3. The humanized chimeric antigen receptor (CAR) of claim 1, wherein thevariable region framework of the anti-CSPG4 binding domain is humanizedmeans that, the heavy chain variable region framework comprises a heavychain FR selected from amino acid sequences shown in SEQ ID NOS: 16-19or any variant thereof; and/or the light chain variable region frameworkcomprises a light chain FR selected from amino acid sequences shown inSEQ ID NOS: 20-23 or any variant thereof.
 4. The humanized chimericantigen receptor (CAR) of claim 1, wherein the variable region frameworkof the anti-CSPG4 binding domain is humanized means that, the heavychain variable region framework comprises a heavy chain FR1 selectedfrom the amino acid sequence shown in SEQ ID NO: 16 or any variantthereof, a heavy chain FR2 selected from the amino acid sequence shownin SEQ ID NO: 17 or any variant thereof, a heavy chain FR3 selected fromthe amino acid sequence shown in SEQ ID NO: 18 or any variant thereof, aheavy chain FR4 selected from the amino acid sequence shown in SEQ IDNO: 19 or any variant thereof; and/or a light chain FR1 selected fromthe amino acid sequence shown in SEQ ID NO: 20 or any variant thereof, alight chain FR2 selected from the amino acid sequence shown in SEQ IDNO: 21 or any variant thereof, a light chain FR3 selected from the aminoacid sequence shown in SEQ ID NO: 22 or any variant thereof, a lightchain FR4 selected from the amino acid sequence shown in SEQ ID NO: 23or any variant thereof.
 5. The humanized chimeric antigen receptor (CAR)of claim 1, wherein the anti-CSPG4 binding domain comprises ananti-CSPG4 antibody or antigen binding part comprising a heavy chainCDR1 selected from the amino acid sequence shown in SEQ ID NO: 5 or anyvariant thereof, a heavy chain CDR2 selected from the amino acidsequence shown in SEQ ID NO: 6 or any variant thereof, a heavy chainCDR3 selected from the amino acid sequence shown in SEQ ID NO: 7 or anyvariant thereof; and/or a light chain CDR1 selected from the amino acidsequence shown in SEQ ID NO: 10 or any variant thereof, a light chainCDR2 selected from the amino acid sequence shown in SEQ ID NO: 11 or anyvariant thereof, a light chain CDR3 selected from the amino acidsequence shown in SEQ ID NO: 12 or any variant thereof.
 6. The humanizedchimeric antigen receptor (CAR) of claim 1, the anti-CSPG4 bindingdomain comprising an anti-CSPG4 antibody or antigen binding partcomprising a heavy chain variable region sequence selected from theamino acid sequence shown in SEQ ID NO: 4 or any variant thereof; and/ora light chain variable region sequence selected from the amino acidsequence shown in SEQ ID NO: 9 or any variant thereof.
 7. The humanizedchimeric antigen receptor (CAR) of claim 1, wherein the anti-CSPG4binding domain comprises an anti-CSPG4 single-chain antibody (scFv),preferably the amino acid sequence of the anti-CSPG4 single-chainantibody is selected from amino acid sequences shown in SEQ ID NOS: 2,14 or any variant thereof.
 8. The humanized chimeric antigen receptor(CAR) of claim 7, wherein the heavy and light chains of the anti-CSPG4single-chain antibody are operably linked by a linker, preferably thelinker comprises an amino acid sequence selected from the amino acidsequence shown in SEQ ID NO: 15 or any variant thereof.
 9. The humanizedchimeric antigen receptor (CAR) of claim 1, wherein the transmembranedomain is selected from one or more of the α, β, ζ chain of TCR, CD3γ,CD3δ, CD3ε, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22,CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB, CD152,CD154, PD-1, NKp44, NKp46, and NKG2D transmembrane domains; preferably,the transmembrane domain is selected from one or more of the CD8α, CD8β,CD4, CD45, PD-1, CD154, and CD28 transmembrane domains; preferably, thetransmembrane domain is selected from one or more of the CD8α, CD28transmembrane domains; more preferably, the amino acid sequence of thetransmembrane domain is selected from the amino acid sequence of SEQ IDNO: 27 or any variant thereof; the hinge region is selected from one ormore of a CD8 extracellular hinge region of CD8, an IgG1 Fc CH2CH3 hingeregion, an IgD hinge region, a CD28 extracellular hinge region, an IgG4Fc CH2CH3 hinge region and a CD4 extracellular hinge region; preferably,the hinge region is the CD8α hinge region; more preferably, the aminoacid sequence of the hinge region is selected from the amino acidsequence of SEQ ID NO: 25 or any variant thereof.
 10. The humanizedchimeric antigen receptor (CAR) of claim 1, wherein the signaltransduction domain is selected from one or more of TCRξ, FcRγ, FcRβ,CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278(ICOS), CD66d, DAP10,DAP12, and CD3ζ intracellular signal regions; preferably, the signaltransduction domain is selected from the CD3ζ intracellular signalregion; more preferably, the amino acid sequence of the signaltransduction domain is selected from the amino acid sequence of SEQ IDNO: 33 or any variant thereof; preferably, the signal transductiondomain further comprises one or more costimulatory domains; preferably,the costimulatory domains are selected from one or more of CARD11, CD2,CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, 4-1BB, CD134, CD150,CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, DAP12, LAT, NKD2C,SLP76, TRIM, FcεRIγ, MyD88, 4-1BBL, and 2B4 intracellular signalregions; preferably, the costimulatory domains are selected from the4-1BB, CD134, CD28, and OX40 intracellular signal regions; preferably,the costimulatory domains are selected from one or more of the 4-1BB andCD28 intracellular signal domain; more preferably, the amino acidsequences of the costimulatory domains are selected from the amino acidsequences of SEQ ID NOS: 29, 31 or any variant thereof.
 11. An isolatednucleic acid molecule comprising the polynucleotide sequence encodingthe humanized chimeric antigen receptor (CAR) of claim 1; preferably,the nucleotide sequence of the nucleic acid molecule is selected fromthe nucleotide sequences of SEQ ID NOS: 1, 13 or any variant thereof.12. A nucleic acid construct comprising the nucleic acid molecule ofclaim 11; preferably, the nucleic acid construct is a viral vector; morepreferably, the viral vector is one or more of a retroviral vector, alentiviral vector, an adenoviral vector, and an adeno-associated viralvector.
 13. A virus comprising the nucleic acid molecule comprising thepolynucleotide sequence encoding the humanized chimeric antigen receptor(CAR) of claim 1 or the nucleic acid construct comprising the nucleicacid molecule; preferably, the virus is one or more of a retrovirus, alentivirus, an adenovirus, and an adeno-associated virus.
 14. Uses ofthe humanized chimeric antigen receptor (CAR) of claim 1, the nucleicacid molecule comprising the polynucleotide sequence encoding thehumanized chimeric antigen receptor (CAR), the nucleic acid constructcomprising the nucleic acid molecule, and the virus comprising thenucleic acid molecule or the nucleic acid construct, in the preparationof genetically modified immune cells targeting tumor cells expressingCSPG4.
 15. An isolated host cell expressing the humanized chimericantigen receptor (CAR) of claim 1, or expressing the isolated nucleicacid molecule comprising the polynucleotide sequence encoding thehumanized chimeric antigen receptor (CAR), or expressing the nucleicacid construct comprising the nucleic acid molecule, or expressing thevirus comprising the nucleic acid molecule or the nucleic acidconstruct; preferably, the host cell is a mammalian cell; morepreferably, the host cell is one or more of a T cell, an NK cell, an γδTcell, an NKT cell, a macrophage or cell line, a PG13 cell line, a 293cell line and cell lines derived therefrom; more preferably, the hostcell is a T cell; most preferably, the host cell is a primarily culturedT cell.
 16. The host cell of claim 15, the host cell further expressesother effector molecules, preferably the other effector moleculescomprise one or more of cytokines, chemokines, another chimeric antigenreceptor (CAR), chemokine receptors, siRNA/shRNAs or sgRNAs knockingdown or knocking out PD-1 expression or proteins blocking PD-L1, TCRs,and safety switches; preferably, the cytokine is selected from one ormore of TNF-α, TNF-β, VEGF, TPO, NGF-β, PDGF, TGF-α, TGF-β, IGF-I,IGF-II, EPO, M-CSF, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-25

LIF

FLT-3, interferon, angiostatin, thrombospondin, and endostatin;preferably, the chemokine is selected from one or more of CCL1, CCL11,CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20,CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3,CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL3,CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCL8, XCL1, XCL2, FAM19A1, FAM19A2,FAM19A3, FAM19A4, and FAM19A5; preferably, the chemokine receptor isselected from one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7,CCR8, CCRL1, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CXCR1, and CXCR2;preferably, the safety switch is selected from one or more of HSVTK,VZVTK, iCaspase-9, iCaspase-1, iCaspase-8, truncated EGFR, and RQR8. 17.A pharmaceutical composition comprising the humanized chimeric antigenreceptor (CAR) of claim 1, the nucleic acid molecule comprising thepolynucleotide sequence encoding the humanized chimeric antigen receptor(CAR), the nucleic acid construct comprising the nucleic acid molecule,the virus comprising the nucleic acid molecule or the nucleic acidconstruct and/or the host cell expressing the humanized chimeric antigenreceptor (CAR) of claim 1, and a pharmaceutically acceptable carrier.18. Applications of the humanized chimeric antigen receptor (CAR) ofclaim 1, the nucleic acid molecule comprising the polynucleotidesequence encoding the humanized chimeric antigen receptor (CAR), thenucleic acid construct comprising the nucleic acid molecule, the viruscomprising the nucleic acid molecule or the nucleic acid construct, thehost cell expressing the humanized chimeric antigen receptor (CAR) ofclaim 1 and/or the pharmaceutical composition comprising the humanizedchimeric antigen receptor (CAR) of claim 1 in the preparation of amedicament; preferably, the medicament is used in diagnosing, treatingor preventing cancer-related diseases; preferably, the medicament isused in diagnosing, treating or preventing tumors expressing CSPG4; morepreferably, the medicament is used in diagnosing, treating or preventingone or more selected from brain cancer, breast cancer, head and neckcancer, melanoma, and mesothelioma; more preferably, the brain cancer isselected from one or more of brain glioblastoma, astrocytoma,meningioma, oligodendroglioma, and glioma, the breast cancer is triplenegative breast cancer, the head and neck cancer is head and necksquamous cell carcinoma.